Methods and assays for male sterile watermelon

ABSTRACT

The present disclosure provides watermelon plants with a male sterile phenotype and their progeny. Such plants may comprise an introgressed genomic region associated with a male sterile phenotype. In certain aspects, compositions, including distinct polymorphic molecular markers and methods for producing, breeding, identifying, and selecting plants or germplasm with a male sterile phenotype are provided.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/733,767, filed Jun.8, 2015, which claims the benefit of U.S. Provisional Application Ser.No. 62/018,413, filed Jun. 27, 2014, each of which is incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and morespecifically to methods and compositions for producing watermelon plantswith male sterile phenotypes.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“SEMB:016US_ST25.txt”, which is 20 kilobytes as measured in theMicrosoft Windows operating system and was created on Jun. 8, 2015, isfiled electronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION

Advances in molecular genetics have made it possible to select plantsbased on genetic markers linked to traits of interest, a process calledmarker-assisted selection (MAS). While breeding efforts to date haveprovided a number of useful watermelon lines and varieties withbeneficial traits, there remains a need in the art for selection ofvarieties with further improved traits and methods for their production.In many cases, such efforts have been hampered by difficulties inidentifying and using alleles conferring beneficial traits. Theseefforts can be confounded by the lack of definitive phenotypic assays,and other issues such as epistasis and polygenic or quantitativeinheritance. In the absence of molecular tools such as MAS, it may notbe practical to attempt to produce certain new genotypes of crop plantsdue to such challenges.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods of determining thegenotype associated with a male sterile phenotype of a watermelon plantor part thereof, comprising the steps of: obtaining a sample of materialfrom said plant or part thereof; and detecting in said sample at least afirst polymorphism in or genetically linked to a locus that confers saidmale sterile phenotype comprising loci NW0249314 (SEQ ID NO: 1) andNW0250301 (SEQ ID NO: 78) or within 15 cM thereof. The invention furtherprovides watermelon plants or parts thereof obtained by said methods.

In another aspect, the invention provides methods of identifying awatermelon plant comprising a genotype associated with a male sterilephenotype, comprising the step of detecting in said plant at least afirst polymorphism in or genetically linked to a locus that confers saidmale sterile phenotype comprising loci NW0249314 (SEQ ID NO: 1) andNW0250301 (SEQ ID NO: 78) on LG2. In some embodiments, said methodsfurther comprise the step of selecting said watermelon plant from apopulation of watermelon plants based on the presence of saidpolymorphism. In certain embodiments, the polymorphism is selected fromthe group consisting of: NW0249314 (SEQ ID NO: 1), NW0249599 (SEQ ID NO:6), NW0250496 (SEQ ID NO: 7), NW0249312 (SEQ ID NO: 12), NW0251153 (SEQID NO: 17), NW0248249 (SEQ ID NO: 22), NW0251130 (SEQ ID NO: 23),NCLAN009404570 (SEQ ID NO: 28), NCLAN009584571 (SEQ ID NO: 33),NCLAN009490296 (SEQ ID NO: 38), NCLAN009490491 (SEQ ID NO: 43),NCLAN009491448 (SEQ ID NO: 48), NW0251340 (SEQ ID NO: 53),NCLAN009490864 (SEQ ID NO: 54), NCLAN009405170 (SEQ ID NO: 59),NW0248760 (SEQ ID NO: 64), NCLAN009104771 (SEQ ID NO: 65), NW0248489(SEQ ID NO: 70), NW0249128 (SEQ ID NO: 71), NW0251464 (SEQ ID NO: 72),NW0248953 (SEQ ID NO: 73), and NW0250301 (SEQ ID NO: 78). In furtherembodiments, the polymorphism is further defined as located in a genomicregion flanked by: loci NCLAN009404570 (SEQ ID NO: 28) andNCLAN009405170 (SEQ ID NO: 59) on LG2. In yet further embodiments, thepolymorphism is NCLAN009584571 (SEQ ID NO: 33). The invention furtherprovides watermelon plants or parts thereof obtained by said methods.

In one aspect, the invention provides methods for producing a watermelonplant that comprises in its genome at least one locus associated with amale sterile phenotype, the method comprising: (i) crossing a firstwatermelon plant and a second watermelon plant at least one of whichcomprises a locus associated with a male sterile phenotype defined by:loci NW0249314 (SEQ ID NO: 1) and NW0250301 (SEQ ID NO: 78) on LG2, orwithin 15 cM thereof; (ii) detecting at least a first polymorphism in orgenetically linked to said locus associated with a male sterilephenotype; and (iii) selecting a watermelon plant based on the presenceof said polymorphism. In certain embodiments, said methods furthercomprise the step of (iv) crossing the watermelon plant of step (iii)with itself or another watermelon plant to produce a further generation.In further embodiments, said methods further comprise the step of: (v)selecting a watermelon plant from the further generation based on thepresence of said polymorphism. In yet further embodiments, steps(iii)-(v) of said methods are repeated from about 3 times to about 10times. In certain embodiments, said polymorphism is selected from thegroup consisting of: NW0249314 (SEQ ID NO: 1), NW0249599 (SEQ ID NO: 6),NW0250496 (SEQ ID NO: 7), NW0249312 (SEQ ID NO: 12), NW0251153 (SEQ IDNO: 17), NW0248249 (SEQ ID NO: 22), NW0251130 (SEQ ID NO: 23),NCLAN009404570 (SEQ ID NO: 28), NCLAN009584571 (SEQ ID NO: 33),NCLAN009490296 (SEQ ID NO: 38), NCLAN009490491 (SEQ ID NO: 43),NCLAN009491448 (SEQ ID NO: 48), NW0251340 (SEQ ID NO: 53),NCLAN009490864 (SEQ ID NO: 54), NCLAN009405170 (SEQ ID NO: 59),NW0248760 (SEQ ID NO: 64), NCLAN009104771 (SEQ ID NO: 65), NW0248489(SEQ ID NO: 70), NW0249128 (SEQ ID NO: 71), NW0251464 (SEQ ID NO: 72),NW0248953 (SEQ ID NO: 73), and NW0250301 (SEQ ID NO: 78). In furtherembodiments, said polymorphism is located in or genetically linked to agenomic region defined by: loci NCLAN009404570 (SEQ ID NO: 28) andNCLAN009405170 (SEQ ID NO: 59) on LG2; or within 15 cM thereof. In yetfurther embodiments, said polymorphism is NCLAN009584571 (SEQ ID NO:33). The invention further provides watermelon plants or parts thereofobtained by said methods.

In a further aspect, the invention provides methods of introgressing anallele into a watermelon plant, the method comprising: (i) genotyping atleast one watermelon plant in a population with respect to at least onepolymorphism located in or genetically linked to a genomic regiondefined by: loci NW0249314 (SEQ ID NO: 1) and NW0250301 (SEQ ID NO: 78)on LG2, or within 15 cM thereof; and (ii) selecting from the populationat least one watermelon plant comprising at least one allele associatedwith a male sterile phenotype. In certain embodiments, said polymorphismis selected from the group consisting of: NW0249314 (SEQ ID NO: 1),NW0249599 (SEQ ID NO: 6), NW0250496 (SEQ ID NO: 7), NW0249312 (SEQ IDNO: 12), NW0251153 (SEQ ID NO: 17), NW0248249 (SEQ ID NO: 22), NW0251130(SEQ ID NO: 23), NCLAN009404570 (SEQ ID NO: 28), NCLAN009584571 (SEQ IDNO: 33), NCLAN009490296 (SEQ ID NO: 38), NCLAN009490491 (SEQ ID NO: 43),NCLAN009491448 (SEQ ID NO: 48), NW0251340 (SEQ ID NO: 53),NCLAN009490864 (SEQ ID NO: 54), NCLAN009405170 (SEQ ID NO: 59),NW0248760 (SEQ ID NO: 64), NCLAN009104771 (SEQ ID NO: 65), NW0248489(SEQ ID NO: 70), NW0249128 (SEQ ID NO: 71), NW0251464 (SEQ ID NO: 72),NW0248953 (SEQ ID NO: 73), and NW0250301 (SEQ ID NO: 78). In furtherembodiments, said polymorphism is located in a genomic region flankedby: loci NCLAN009404570 (SEQ ID NO: 28) and NCLAN009405170 (SEQ ID NO:59) on LG2; or within 15 cM thereof. In yet further embodiments, thepolymorphism is NCLAN009584571 (SEQ ID NO: 33). The invention furtherprovides watermelon plants or parts thereof obtained by said methods.

In yet another aspect, the invention provides a probe or primer sequencefor detecting the presence of an allele in a watermelon plant, saidmarker comprising a sequence selected from the group consisting of:NW0249314 (SEQ ID NO: 1), NW0249599 (SEQ ID NO: 6), NW0250496 (SEQ IDNO: 7), NW0249312 (SEQ ID NO: 12), NW0251153 (SEQ ID NO: 17), NW0248249(SEQ ID NO: 22), NW0251130 (SEQ ID NO: 23), NCLAN009404570 (SEQ ID NO:28), NCLAN009584571 (SEQ ID NO: 33), NCLAN009490296 (SEQ ID NO: 38),NCLAN009490491 (SEQ ID NO: 43), NCLAN009491448 (SEQ ID NO: 48),NW0251340 (SEQ ID NO: 53), NCLAN009490864 (SEQ ID NO: 54),NCLAN009405170 (SEQ ID NO: 59), NW0248760 (SEQ ID NO: 64),NCLAN009104771 (SEQ ID NO: 65), NW0248489 (SEQ ID NO: 70), NW0249128(SEQ ID NO: 71), NW0251464 (SEQ ID NO: 72), NW0248953 (SEQ ID NO: 73),and NW0250301 (SEQ ID NO: 78).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows data for markers in the interval of NW0249314/NW0249599and NW0250301. Germplasm shown represents diverse watermelon breedinglines carrying the ms-1 introgression for which male sterility ismaintained. A region of heterozygosity was identified for some of theselines near the SNP markers NW0248249 (SEQ ID NO: 22), NW0251340 (SEQ IDNO: 53), and NW0248760 (SEQ ID NO: 64). The markers shaded in gray arethe indicated region of ms-1 gene as estimated from inference of geneticdistance on the consensus map. The marker alleles shaded in gray are theindicated heterozygotic condition of the two marker alleles at thatmarker locus.

FIG. 2: Shows genotype and phenotype data for representative lines fromfine mapping populations. Gray shading denotes fertile, no shadingdenotes sterile.

FIG. 3: Shows a sequence alignment for two sterile families(WAS-45-2158S and WML-45-1445) showing the 10 bp deletion in the sterilelines assayed by marker NCLAN009584571 (SEQ ID NO: 33).

DETAILED DESCRIPTION OF THE INVENTION

Hybrid watermelon varieties typically exhibit higher yield, higherquality, and more uniform fruit size than open pollinated varieties.Commercial watermelon hybrid varieties include both diploids andtriploids. A diploid hybrid is made by crossing two inbred diploidparent lines. A triploid hybrid is made by crossing a tetraploid femaleparent line with a diploid male parent line. Controlled pollination isessential to hybrid seed production. The production of hybrid seed bycontrolled pollination requires that both male and female flower budsare protected and that female flowers are hand-pollinated. Controlledpollination is labor-intensive and the cost of hybrid seed can be 10-100times that of open-pollinated seed. The development and use of malesterile watermelon varieties can greatly reduce the cost of hybrid seedproduction as the pollen production in the female parent line iseliminated. The use of a male sterile line eliminates the need forcontrolled pollination.

Male sterility is the failure of plants to produce functional anthers,pollen, or male gametes. Male sterility facilitates cross hybridizationand eliminates the need for laborious and costly hand emasculation andpollination. Several male sterile genes have been identified inwatermelon, including the ms-1 gene. The ms-1 nuclear gene controls malesterility and, in plants with an ms-1 introgression, the normaldevelopment of anthers is hindered while female flower development isnormal. The gene eliminates pollen production and results in an idealfemale parent line for hybrid production.

The invention overcomes limitations in the prior art by providingmethods and compositions for introgressing an ms-1 recessive allele intoelite watermelon lines, permitting efficient production of hybrid seed.Commercial watermelon hybrids can be diploid and triploid. The diploidhybrids are produced from two diploid inbred parent lines while thetriploid hybrids use a female tetraploid line and a male diploid parentline. The ms-1 recessive allele can be introgressed into the femalediploid or tetraploid line.

The present invention provides compositions and methods for theintrogression of the ms-1 allele into elite watermelon lines. Themethods and compositions allow the use of marker assisted selection(MAS). In particular, genomic regions, alleles, polymorphic nucleicacids, and linked markers have been identified that are associated withmale sterility in watermelon. A genetic map of the ms-1 gene wasconstructed. In one example, a segregating population from the cross ofWSB-45-101S×SVR14227703 was developed, and phenotypic and genotypic datafor 250 F2 individuals was collected. A linkage map was constructed,which consisted of 240 SNP markers and had a total of 20 linkage groupsand total length of 1514.3 cM. A genomic region associated with a malesterile phenotype was identified at watermelon linkage group 2, betweenSNP markers NW0249314 (SEQ ID NO:1)/NW0249599 (SEQ ID NO: 6)(colocalized) and NW0250301 (SEQ ID NO: 78). NW0250301 is the mostclosely linked marker located 16.3 cM downstream of the ms-1 gene. In afurther example, the ms-1 gene was mapped on linkage group 2 within theinterval of the SNP markers NCLAN009404570 (SEQ ID NO: 28) andNCLAN009405170 (SEQ ID NO: 59), corresponding to about 150 kb onchromosome 6.

The present invention represents a significant advantage by providinggenomic regions, alleles, polymorphic nucleic acids, and linked markersthat are associated with male sterility in watermelon, methods of usingsuch for the production or identification of watermelon plantscomprising male sterility and watermelon plants produced by the methodswherein the watermelon plants comprise at least a first introgressedlocus conferring male sterility. In accordance with the invention, theintrogressed locus may be newly introgressed into a given genomicbackground of a specific variety or cultivar. Certain embodimentsprovide methods of detecting in a watermelon plant a genotype associatedwith male sterility. Other embodiments provide methods of identifyingand selecting a watermelon plant comprising in its genome a genotypeassociated with male sterility. Further embodiments provide methods ofproducing a watermelon plant that comprises in its genome at least oneintrogressed locus associated with male sterility and methods forintrogressing such an allele into a watermelon plant. Watermelon plantsand parts thereof made by any of said methods are also provided for, aswell as polymorphic nucleic acid sequences that may be used in theproduction and identification of such plants.

The markers associated with male sterility provided herein are usefulfor a variety of methods which utilize genotyping; the availability ofmarkers reduce costs and unreliability associated with phenotypingassays. As an example, seeds or seedlings may be tested for the presenceof marker alleles associated with the male sterility phenotype prior tosowing or transplanting, allowing selection and planting ortransplanting of only male sterile seeds or seedlings for the femaleparent of a hybrid. The use of the markers disclosed herein results inseed production with up to 100% hybrid seed by reducing the risk of selfpollination in a production field and also reduces or eliminates thepresence of parent (non-hybrid) seed in a seed bag. Additionally, use ofthese markers can improve production capacity in a field. Planting onlymale sterile seed or plants of the female hybrid parent line providesimprovement over the current method which includes planting, waitinguntil flowers develop and then passing over the field to rogue fertilefemale plants. The elimination of fertile female plants decreases laborinput and resource costs. Further, breeding programs can be designed tospecifically drive the frequency of specific favorable phenotypes bytargeting particular genotypes. Fidelity of these associations may bemonitored continuously to maintain predictive ability and informedbreeding decisions. For example, markers may be used to identify andselect the heterozygous condition of the ms-1 locus in progeny of across so that the trait can be conferred through a pedigree.

In accordance with the invention, one of skill in the art may identify acandidate germplasm source possessing male sterility as describedherein, but which is lacking one or more traits which the plant breederseeks to have in a variety or parent line thereof. The techniques of theinvention may be used to identify male sterile phenotypes by utilizinggenetic markers associated with the phenotype. Alternatively, suchtechniques may employ phenotypic assays to identify desired plantseither alone or in combination with genetic assays.

Markers, including those described herein, may be assayed through theuse of an automated, high-throughput, nondestructive seed samplingmethod as, for example, described in U.S. Pat. No. 8,076,076,incorporated herein by reference in its entirety. Briefly, the methodcomprises providing a population of seeds, removing a tissue samplecomprising cells with nucleic acids from each seed in the population,analyzing the nucleic acids extracted from each seed for the presence ofat least one genetic marker indicating the presence of a male-sterilegene, selecting seeds from the population based upon the presence of themale-sterile marker, and cultivating a fertile plant from the seed. Themethod additionally includes receiving and orienting the individualseeds, while preserving the germination viability of each seed, at anorientation subsystem of the automated seed processing system. Themethod further includes removing a tissue sample from the individualseeds, while preserving the germination viability of each seed, at asampling subsystem of the automated seed processing system. Stillfurther, the method includes depositing each seed into a selected wellin a selected one of a plurality of seed collection trays after the seedhas had a tissue sample removed and depositing each tissue sample into aselected well in a selected one of a plurality of sample collectiontrays, utilizing a seed and sample transport subsystem of the automatedseed processing system. The method further includes storing in adatabase the selected well in the selected sample collection tray intowhich each sample was deposited and the selected well in the selectedseed collection tray into which each corresponding sampled seed wasdeposited, such that each sample and corresponding seed from which thesample was removed can be tracked to pre-select seeds of interest.

Generally, watermelon sex expression can be divided into qualitativecategories as being monoecious (separate male and female flowers on thesame plant), andromonoecious (separate male and hermaphrodite flowers onthe same plant), and trimonoecious (separate male, female andhermaphrodite flowers on the same plant).

In accordance with the invention, male sterility refers to the failureof plants to produce functional anthers, pollen, or male gametes, and/orany and all combinations thereof that one or more breeder, grower, orconsumer may find advantageous for certain applications. Several malesterility genes have been identified in watermelon, including the ms-1gene. The male sterility trait has been shown to be controlled by thegenomic regions identified herein, and this trait may be introgressedinto desired genetic backgrounds using the methods of the invention.

The present invention relates to watermelon genomic regions, polymorphicnucleic acids, and alleles associated with male sterility. This trait isimportant during the production of hybrid watermelon plants, which areproduced by crossing a seed parent plant with a pollen parent plant.Typically, a female inbred parent line and a pollen donor (male) inbredline are crossed to produce a given commercial hybrid. For example,hybrid watermelon can be produced by crossing a diploid parent plantwith a tetraploid parent plant or by crossing two diploid parent plants.Male sterility is important for hybrid seed production as it avoids theneed for controlled pollination.

The genomic regions, polymorphic nucleic acids, and alleles of thepresent invention allow production of watermelon plants with decreasedfrequency of anthers on female flowers of seed parent inbred plantsduring hybrid seed production, thereby resulting in an increase inpercent hybridity of the produced seed (less selfed seed). The presentinvention therefore relates to markers and genomic regions linked tomale sterility and methods of use thereof in order to select parentlines that would better serve as seed parents for hybrid production.

The invention provides for the introgression of at least a first locusconferring male sterility into a given genetic background. Successfulwatermelon production depends on attention to various horticulturalpractices. These include soil management with special attention toproper fertilization, crop establishment with appropriate spacing, weedcontrol, the introduction of bees or other insects for pollination,irrigation, pest management, and, if producing fruit from triploidplants, a suitable pollen source for producing seedless (triploid)watermelon. Watermelon flower size and shape; rind color, thickness andtoughness; sex expression; flesh color, texture, and sugar content; andfreedom from fruit defects are all important characteristics to beconsidered in selection of watermelon varieties.

Watermelon crops can be established from seed or from transplants.Transplanting can result in an earlier crop compared with a cropproduced from direct seeding. When a grower wants to raise a seedlessfruited crop, transplanting can be preferred. Transplanting helpsachieve complete plant stands rapidly, especially where higher seedcosts, as with triploid seeds, make direct-seeding risky.

Watermelon breeders are challenged with anticipating changes in growingconditions, new pathogen pressure, and changing consumer preferences.With these projections, a breeder will attempt to create new cultivarsthat will fit the needs of growers, shippers, retailers, and consumers.Thus, the breeder is challenged to combine, in a single genotype, asmany favorable attributes as possible.

Genomic Region, Polymorphic Nucleic Acids, and Alleles Associated withMale Sterility in Watermelon

Applicants have identified genomic regions, alleles, polymorphic nucleicacids, linked markers, and the like that when present in certain allelicforms are associated with watermelon male sterility.

A genomic region associated with a male sterile phenotype was identifiedat watermelon linkage group 2, flanked by loci NW0249314 (SEQ ID NO:1)/NW0249599 (SEQ ID NO: 6 (colocalized) and NW0250301 (SEQ ID NO: 78).Another genomic region associated with a male sterile phenotype wasidentified at watermelon linkage group 2 (LG2), flanked by lociNCLAN009404570 (SEQ ID NO: 28) and NCLAN009405170 (SEQ ID NO: 59).

Certain of the various embodiments of the present disclosure thusutilize one or more polymorphic nucleic acid markers or alleles locatedin one or more of these regions or subregions on LG2. For example,NW0250301 (SEQ ID NO: 78) is a closely linked marker located 16.3 cMdownstream of the ms-1 gene.

The above markers and allelic states are exemplary. One of skill in theart would recognize how to identify watermelon plants with otherpolymorphic nucleic acid markers and allelic states thereof related towatermelon male sterility consistent with the present disclosure. One ofskill in the art would also know how to identify the allelic state ofother polymorphic nucleic acid markers located in the genomic region(s)or linked to the QTL or other markers identified herein, to determinetheir association with watermelon male sterility.

Watermelons are natural diploids, having their chromosomes arranged inpairs. Watermelon plants, however, can undergo a duplication of theirentire set of chromosomes and exist as tetraploids. While it is uncommonfor watermelons to produce spontaneous tetraploids, this process can beroutinely produced in the laboratory using cell biology techniques.Triploid seeds can be produced by crossing a tetraploid parent by adiploid parent. When triploid plants are grown, seed formation in thefruit aborts because of the ploidy level differences, resulting inseedless fruits.

In certain embodiments of methods of the invention, a diploid (female)parent plant is homozygous for a polymorphic nucleic acid marker alleleassociated with the male sterile phenotype. The diploid (female) parentis crossed with another diploid parent lacking the polymorphic nucleicacid marker allele associated with the male sterile phenotype to producediploid hybrid progeny. This results in one copy of the polymorphicmarker allele associated with the male sterile phenotype (from thediploid female parent) and one allele not associated with the malesterile phenotype (from the diploid male parent) in the diploid hybrid.Alternatively, in certain embodiments of methods of the invention, atetraploid parent plant is homozygous for a polymorphic nucleic acidmarker allele associated with the male sterile phenotype. The tetraploidparent is crossed with a diploid lacking the polymorphic nucleic acidmarker allele associated with the male sterile phenotype, to producetriploid hybrid progeny. This results in two copies of the polymorphicmarker allele associated with the male sterile phenotype (from thetetraploid parent) and one allele not associated with the male sterilephenotype (from the diploid parent) in the triploid hybrid.

Certain embodiments of the invention contemplate the use ofdihaploidization to produce an inbred line. A haploid plant has only onecopy of each chromosome instead of the normal pair of chromosomes in adiploid plant. Haploid plants can be produced, for example, by treatingwith a haploid inducer. Haploid plants can be subjected to treatmentthat causes the single copy chromosome set to double, producing aduplicate copy of the original set. The resulting plant is termed a“double-haploid” and contains pairs of chromosomes that are generally ina homozygous allelic state at any given locus. Dihaploidization canreduce the time required to develop new inbred lines in comparison todeveloping lines through successive rounds of backcrossing.

One of skill in the art would understand that polymorphic nucleic acidsthat are located in the genomic regions identified herein may be used incertain embodiments of the methods of the invention. Given the genomicregions and polymorphic markers identified herein, additional markerslocated either within or near a genomic region described herein that areassociated with the phenotype can be obtained by typing new markers invarious germplasm. The genomic region and polymorphic markers identifiedherein can also be mapped relative to any publicly available physical orgenetic map to place the region described herein on such map. One ofskill in the art would also understand that additional polymorphicnucleic acids that are genetically linked to the genomic regionassociated with a male sterile phenotype and that map within 40 cM, 20cM, 10 cM, 5 cM, or 1 cM of the genomic region or the markers associatedwith a male sterile phenotype may also be used.

Introgression of a Genomic Locus Associated with Male Sterility

Marker-assisted introgression involves the transfer of a chromosomalregion defined by one or more markers from a first germplasm to a secondgermplasm. Offspring of a cross that contain the introgressed genomicregion can be identified by the combination of markers characteristic ofthe desired introgressed genomic region from a first germplasm (e.g.male sterile phenotype germplasm) and both linked and unlinked markerscharacteristic of the desired genetic background of a second germplasm.

Flanking markers that identify a genomic region associated with adesired sex expression phenotype can include any loci described hereinon linkage group 2 (LG2), and those that identify sub-regions thereofcan include any loci or loci intervals described herein on LG2.

For example, flanking markers that identify a genomic region orsubregion include those defined by loci NW0249314 (SEQ ID NO: 1) andNW0250301 (SEQ ID NO: 78) on LG2 or within 15 cM thereof, or defined byloci NCLAN009404570 (SEQ ID NO: 28) and NCLAN009405170 (SEQ ID NO: 59)on LG2 or within 15 cM thereof.

In further embodiments, markers associated with a male sterile phenotypeare: NW0249314 (SEQ ID NO: 1) and NW0250301 (SEQ ID NO: 78) on LG2 orflanked by loci NCLAN009404570 (SEQ ID NO: 28) and NCLAN009405170 (SEQID NO: 59) on LG2. In further embodiments, markers include NW0249314(SEQ ID NO: 1), NW0249599 (SEQ ID NO: 6), NW0250496 (SEQ ID NO: 7),NW0249312 (SEQ ID NO: 12), NW0251153 (SEQ ID NO: 17), NW0248249 (SEQ IDNO: 22), NW0251130 (SEQ ID NO: 23), NCLAN009404570 (SEQ ID NO: 28),NCLAN009584571 (SEQ ID NO: 33), NCLAN009490296 (SEQ ID NO: 38),NCLAN009490491 (SEQ ID NO: 43), NCLAN009491448 (SEQ ID NO: 48),NW0251340 (SEQ ID NO: 53), NCLAN009490864 (SEQ ID NO: 54),NCLAN009405170 (SEQ ID NO: 59), NW0248760 (SEQ ID NO: 64),NCLAN009104771 (SEQ ID NO: 65), NW0248489 (SEQ ID NO: 70), NW0249128(SEQ ID NO: 71), NW0251464 (SEQ ID NO: 72), NW0248953 (SEQ ID NO: 73),or NW0250301 (SEQ ID NO: 78). In certain embodiments, the marker isNCLAN009584571 (SEQ ID NO: 33). Exemplary markers provided by theinvention are shown in Table 1.

TABLE 1 Markers associated with male sterility in watermelon. Allele ofmale DNA Probe VIC Probe FAM Primer F Primer R Genetic Allele of sterileSequence Sequence Sequence Sequence Sequence Position WT/ parent/(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID Marker LG (cM) fertile phenotypeNO) NO) NO) NO) NO) NW0249314 2 39.92 C T  1  2  3  4  5 NW0249599 239.92 C T  6 NW0250496 2 43.44 C T  7  8  9 10 11 NW0249312 2 49.9 G A12 13 14 15 16 NW0251153 2 59.39 C A 17 18 19 20 21 NW0248249 2 73.15 22NW0251130 2 80.02 T A 23 24 25 26 27 NCLAN009404570 2 80.96 G T 28 29 3031 32 NCLAN009584571 2 82.03 GTTTCAGTTC * 33 34 35 36 37(SEQ. ID NO: 83) NCLAN009490296 2 82.05 C G 38 39 40 41 42NCLAN009490491 2 82.05 C T 43 44 45 46 47 NCLAN009491448 2 82.46 G C 4849 50 51 52 NW0251340 2 83 53 NCLAN009490864 2 83.16 G A 54 55 56 57 58NCLAN009405170 2 83.67 G A 59 60 61 62 63 NW0248760 2 100.93 64NCLAN009104771 2 102.4   A G 65 66 67 68 69 NW0248489 2 115.84 C A 70NW0249128 2 120.96 G C 71 NW0251464 2 122.47 G A 72 NW0248953 2 131.69 TA 73 74 75 76 77 NW0250301 2 134.25 78 79 80 81 82

Flanking markers that fall on both the telomere proximal end and thecentromere proximal end of any of these genomic intervals may be usefulin a variety of breeding efforts that include, but are not limited to,introgression of genomic regions associated with a male sterilephenotype into a genetic background comprising markers associated withgermplasm that ordinarily contains a genotype associated with anotherphenotype.

Markers that are linked and either immediately adjacent or adjacent tothe identified male sterile phenotype genomic region that permitintrogression of the genomic region in the absence of extraneous linkedDNA from the source germplasm containing the genomic region are providedherein. Those of skill in the art will appreciate that when seeking tointrogress a smaller genomic region associated with a male sterilephenotype described herein, that any of the telomere proximal orcentromere proximal markers that are immediately adjacent to a largergenomic region associated with a male sterile phenotype can be used tointrogress that smaller genomic region.

A marker within about 40 cM of a marker associated with a male sterilephenotype described herein may be useful in a variety of breedingefforts that include, but are not limited to, introgression of genomicregions associated with a male sterile phenotype into a geneticbackground comprising markers associated with germplasm that ordinarilycontains a genotype associated with another phenotype. For example, amarker within 40 cM, 20 cM, 15 cM, 10 cM, 5 cM, 2 cM, or 1 cM of amarker associated with a male sterile phenotype or marker describedherein can be used for marker-assisted introgression of a male sterilephenotype.

A marker within about 40 cM of a male sterile phenotype marker on LG2described herein can be used for marker-assisted introgression of a malesterile phenotype. As described above, a male sterile phenotype markeron LG2 can include NW0250301 (SEQ ID NO: 78).

Watermelon plants or germplasm comprising an introgressed region that isassociated with a male sterile phenotype wherein at least 10%, 25%, 50%,75%, 90%, or 99% of the remaining genomic sequences carry markerscharacteristic of plant or germplasm that otherwise or ordinarilycomprise a genomic region associated with another phenotype, are thusprovided. Furthermore, watermelon plants comprising an introgressedregion where closely linked regions adjacent and/or immediately adjacentto the genomic regions, QTL, and markers provided herewith that comprisegenomic sequences carrying markers characteristic of watermelon plantsor germplasm that otherwise or ordinarily comprise a genomic regionassociated with the phenotype are also provided.

Development of Male Sterile Watermelon Varieties

Male sterility in watermelon is important in terms of production of F₁hybrid plants, and has significance to growers, processors, retailers,and customers. The present invention discloses the identification ofgenomic regions and alleles associated with male sterility, as well assingle nucleotide polymorphism (SNP) markers genetically linked to andpredictive of such loci that can be used for the tracking andintrogression of male sterile traits into germplasm, such as bymarker-assisted selection and/or marker-assisted backcrossing.

The invention thus allows the tracking and introduction of any of thegenetic regions or markers identified herein into a given geneticbackground. One of ordinary skill will understand that a male sterilephenotype can be introgressed from one genotype to another using aprimary locus described herein via marker assisted selection.Accordingly, a germplasm source can be selected that has a male sterilephenotype. A breeder can use the markers identified herein to select formale sterility or track male sterility during breeding using markerassisted selection for the region described herein. Provided with thepresent disclosure, one of ordinary skill can introduce male sterilityinto any genetic background.

For example, using the markers described herein, seed can be genotypedfor the ms-1 markers of the present invention in order to select sterileplants for hybrid seed production. This method yields 100% hybridity inseed lot production, and reduces or eliminates parent (non-hybrid) seedcontamination. This results in improved production capacity in thefield, and represents an improvement over conventional methods ofplanting which involve selection of plants based on phenotype androguing of fertile plants. Moreover, marker-assisted selection allowsselection of the heterozygous state for breeding.

Thus, the genetic regions and markers identified herein can be used formarker assisted selection for male sterility in watermelon. Thisdiscovery of male sterility markers will facilitate the development ofwatermelon having a male sterile phenotype.

For most breeding objectives, commercial breeders work within germplasmthat is “cultivated type” or “elite.” This germplasm is easier to breedbecause it generally performs well when evaluated for horticulturalperformance. However, the performance advantage a cultivated germplasmprovides is sometimes offset by a lack of allelic diversity. Breedersgenerally accept this tradeoff because progress is faster when workingwith cultivated material than when breeding with genetically diversesources.

In contrast, when a breeder makes either intra-specific crosses, orinter-specific crosses, a converse trade off occurs. In these examples,a breeder typically crosses cultivated germplasm with a non-cultivatedtype. In such crosses, the breeder can gain access to novel alleles fromthe non-cultivated type, but may have to overcome the genetic dragassociated with the donor parent. Because of the difficulty with thisbreeding strategy, this approach often fails because of fertility andfecundity problems. The difficulty with this breeding approach extendsto many crops, and is exemplified with an important disease resistantphenotype that was first described in tomato in 1944 (Smith, Proc. Am.Soc. Hort. Sci. 44:413-16). In this cross, a nematode disease resistancewas transferred from L. peruvianum (PI128657) into a cultivated tomato.Despite intensive breeding, it was not until the mid-1970s that breederscould overcome the genetic drag and release successful lines carryingthis trait. Even today tomato breeders deliver this disease resistancegene to a hybrid variety from only one parent.

In watermelon, the plant introduction (PI) accessions are typicallylines that produce fruits with undesirable production and eatingqualities. Even though these lines have poor horticultural qualities,some watermelon breeders attempt to breed with these PI lines becausethey can potentially contain novel alleles. The process of introgressingnovel resistance genes from the PI lines into acceptable commercialtypes is a long and often arduous process. This process can be difficultbecause the trait may be polygenic, or have low heritability, or havelinkage drag or some combination thereof.

Some phenotypes are determined by the genotype at one locus. Thesesimple traits, like those studied by Gregor Mendel, fall indiscontinuous categories such as green or yellow seeds. Other variationobserved in nature, however, is continuous, like yield in field corn, orhuman blood pressure. Unlike simply inherited traits, continuousvariation can be the result of polygenic inheritance. Loci that affectcontinuous variation are referred to as QTLs. Variation in the phenotypeof a quantitative trait is the result of the allelic composition at theQTLs and the environmental effect. The heritability of a trait is theproportion of the phenotypic variation attributed to the geneticvariance. This ratio varies between 0 and 1.0. Thus, a trait withheritability near 1.0 is not greatly affected by the environment. Thoseskilled in the art recognize the importance of creating commercial lineswith high heritability horticultural traits because these cultivars willallow growers to produce a crop with uniform market specifications.

Molecular Assisted Breeding Techniques

Genetic markers that can be used in the practice of the presentinvention include, but are not limited to, Restriction Fragment LengthPolymorphisms (RFLP), Amplified Fragment Length Polymorphisms (AFLP),Simple Sequence Repeats (SSR), simple sequence length polymorphisms(SSLPs), Single Nucleotide Polymorphisms (SNP), Insertion/DeletionPolymorphisms (Indels), Variable Number Tandem Repeats (VNTR), andRandom Amplified Polymorphic DNA (RAPD), isozymes, and others known tothose skilled in the art. Marker discovery and development in cropsprovides the initial framework for applications to marker-assistedbreeding activities (U.S. Patent Pub. Nos.: 2005/0204780, 2005/0216545,2005/0218305, and 2006/00504538). The resulting “genetic map” is therepresentation of the relative position of characterized loci(polymorphic nucleic acid markers or any other locus for which allelescan be identified) to each other.

Polymorphisms comprising as little as a single nucleotide change can beassayed in a number of ways. For example, detection can be made byelectrophoretic techniques including a single strand conformationalpolymorphism (Orita et al. (1989) Genomics, 8(2), 271-278), denaturinggradient gel electrophoresis (Myers (1985) EPO 0273085), or cleavagefragment length polymorphisms (Life Technologies, Inc., Gathersberg, Md.20877), but the widespread availability of DNA sequencing machines oftenmakes it easier to simply sequence amplified products directly. Once thepolymorphic sequence difference is known, rapid assays can be designedfor progeny testing, typically involving some version of PCRamplification of specific alleles (PASA, Sommer, et al. (1992)Biotechniques 12(1), 82-87), or PCR amplification of multiple specificalleles (PAMSA, Dutton and Sommer (1991) Biotechniques, 11(6),700-7002).

As a set, polymorphic markers serve as useful tools for assaying plantsto determine the degree of identity of lines or varieties (U.S. Pat. No.6,207,367). These markers form the basis for determining associationswith phenotypes and can be used to drive genetic gain. In certainembodiments of methods of the invention, polymorphic nucleic acids canbe used to detect in a watermelon plant a genotype associated with malesterility, identify a watermelon plant with a genotype associated withmale sterility, and to select a watermelon plant with a genotypeassociated with male sterility. In certain embodiments of methods of theinvention, polymorphic nucleic acids can be used to produce a watermelonplant that comprises in its genome an introgressed locus associated withmale sterility. In certain embodiments of the invention, polymorphicnucleic acids can be used to breed progeny watermelon plants comprisinga locus associated with male sterility.

Certain genetic markers may include “dominant” or “codominant” markers.“Codominant” markers reveal the presence of two or more alleles (two perdiploid individual). “Dominant” markers reveal the presence of only asingle allele. Markers are preferably inherited in codominant fashion sothat the presence of both alleles at a diploid locus, or multiplealleles in triploid or tetraploid loci, are readily detectable, and theyare free of environmental variation, i.e., their heritability is 1. Amarker genotype typically comprises two marker alleles at each locus ina diploid organism. The marker allelic composition of each locus can beeither homozygous or heterozygous. Homozygosity is a condition whereboth alleles at a locus are characterized by the same nucleotidesequence. Heterozygosity refers to different conditions of the allele ata locus.

Nucleic acid-based analyses for determining the presence or absence ofthe genetic polymorphism (i.e. for genotyping) can be used in breedingprograms for identification, selection, introgression, and the like. Awide variety of genetic markers for the analysis of geneticpolymorphisms are available and known to those of skill in the art. Theanalysis may be used to select for genes, portions of genes, QTL,alleles, or genomic regions that comprise or are linked to a geneticmarker that is linked to or associated with a male sterile phenotype.

As used herein, nucleic acid analysis methods include, but are notlimited to, PCR-based detection methods (for example, TaqMan assays),microarray methods, mass spectrometry-based methods and/or nucleic acidsequencing methods, including whole genome sequencing. In certainembodiments, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

One method of achieving such amplification employs the polymerase chainreaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol.51:263-273; European Patent 50,424; European Patent 84,796; EuropeanPatent 258,017; European Patent 237,362; European Patent 201,184; U.S.Pat. Nos. 4,683,202; 4,582,788; and 4,683,194), using primer pairs thatare capable of hybridizing to the proximal sequences that define apolymorphism in its double-stranded form. Methods for typing DNA basedon mass spectrometry can also be used. Such methods are disclosed inU.S. Pat. Nos. 6,613,509 and 6,503,710, and references found therein.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039;7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of whichare incorporated herein by reference in their entirety. However, thecompositions and methods of the present invention can be used inconjunction with any polymorphism typing method to type polymorphisms ingenomic DNA samples. These genomic DNA samples used include but are notlimited to genomic DNA isolated directly from a plant, cloned genomicDNA, or amplified genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods as disclosed in U.S. Pat. No. 5,800,944 where sequence ofinterest is amplified and hybridized to probes followed by ligation todetect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al.,Bioinformatics 21:3852-3858 (2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening ofa plurality of polymorphisms. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Target nucleic acid sequence can also be detected by probe linkingmethods as disclosed in U.S. Pat. No. 5,616,464, employing at least onepair of probes having sequences homologous to adjacent portions of thetarget nucleic acid sequence and having side chains which non-covalentlybind to form a stem upon base pairing of the probes to the targetnucleic acid sequence. At least one of the side chains has aphotoactivatable group which can form a covalent cross-link with theother side chain member of the stem.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extensionof a nucleotide primer that is adjacent to a polymorphism to incorporatea detectable nucleotide residue upon extension of the primer. In certainembodiments, the SBE method uses three synthetic oligonucleotides. Twoof the oligonucleotides serve as PCR primers and are complementary tosequence of the locus of genomic DNA which flanks a region containingthe polymorphism to be assayed. Following amplification of the region ofthe genome containing the polymorphism, the PCR product is mixed withthe third oligonucleotide (called an extension primer) which is designedto hybridize to the amplified DNA adjacent to the polymorphism in thepresence of DNA polymerase and two differentially labeleddideoxynucleosidetriphosphates. If the polymorphism is present on thetemplate, one of the labeled dideoxynucleosidetriphosphates can be addedto the primer in a single base chain extension. The allele present isthen inferred by determining which of the two differential labels wasadded to the extension primer. Homozygous samples will result in onlyone of the two labeled bases being incorporated and thus only one of thetwo labels will be detected. Heterozygous samples have both allelespresent, and will thus direct incorporation of both labels (intodifferent molecules of the extension primer) and thus both labels willbe detected.

In another method for detecting polymorphisms, SNPs and Indels can bedetected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′ 4 3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

In another embodiment, the locus or loci of interest can be directlysequenced using nucleic acid sequencing technologies. Methods fornucleic acid sequencing are known in the art and include technologiesprovided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience(Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-CORBiosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.),Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston,Tex.). Such nucleic acid sequencing technologies comprise formats suchas parallel bead arrays, sequencing by ligation, capillaryelectrophoresis, electronic microchips, “biochips,” microarrays,parallel microchips, and single-molecule arrays.

The markers to be used in the methods of the present invention may bediagnostic of origin in order for inferences to be made about subsequentpopulations. Experience to date suggests that SNP markers may be idealfor mapping because the likelihood that a particular SNP allele isderived from independent origins in the extant populations of aparticular species is very low. As such, SNP markers appear to be usefulfor tracking and assisting introgression of QTLs.

Definitions

The following definitions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which watermelon plantscan be regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

As used herein, the term “male sterility” refers to the failure ofplants to produce functional anthers, pollen, or male gametes, and canbe cytoplasmic, genetic, or both.

As used herein, the term “population” means a genetically heterogeneouscollection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group ofsimilar plants that by their genetic pedigrees and performance can beidentified from other varieties within the same species.

As used herein, an “allele” refers to one of two or more alternativeforms of a genomic sequence at a given locus on a chromosome.

A “Quantitative Trait Locus (QTL)” is a chromosomal location thatencodes for at least a first allele that affects the expressivity of aphenotype.

As used herein, a “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsinclude, but are not limited to, genetic markers, biochemical markers,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, the term “phenotype” means the detectablecharacteristics of a cell or organism that can be influenced by geneexpression.

As used herein, the term “genotype” means the specific allelic makeup ofa plant.

As used herein, the term “introgressed,” when used in reference to agenetic locus, refers to a genetic locus that has been introduced into anew genetic background, such as through backcrossing. Introgression of agenetic locus can thus be achieved through plant breeding methods and/orby molecular genetic methods. Such molecular genetic methods include,but are not limited to, various plant transformation techniques and/ormethods that provide for homologous recombination, non-homologousrecombination, site-specific recombination, and/or genomic modificationsthat provide for locus substitution or locus conversion.

As used herein, the term “linked,” when used in the context of nucleicacid markers and/or genomic regions, means that the markers and/orgenomic regions are located on the same linkage group or chromosome suchthat they tend to segregate together at meiosis.

As used herein, the term “maturity” means maturity of fruit development.Maturity indicates the time a watermelon fruit is ready to be harvested.In watermelon, the maturity comes associated with changes in flesh colorand sugar content.

As used herein, the term “denoting” when used in reference to a plantgenotype refers to any method whereby a plant is indicated to have acertain genotype. This includes any means of identification of a planthaving a certain genotype. Indication of a certain genotype may include,but is not limited to, any entry into any type of written or electronicmedium or database whereby the plant's genotype is provided. Indicationsof a certain genotype may also include, but are not limited to, anymethod where a plant is physically marked or tagged. Illustrativeexamples of physical marking or tags useful in the invention include,but are not limited to, a barcode, a radio-frequency identification(RFID), a label, or the like.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

EXAMPLES

The following disclosed embodiments are merely representative of theinvention which may be embodied in various forms. Thus, specificstructural, functional, and procedural details disclosed in thefollowing examples are not to be interpreted as limiting.

Example 1

A segregating population was developed from a cross ofWSB-45-101S×SVR14227703 and was used for mapping of the ms-1 gene;WSB-45-101S is a diploid Sugar Baby-type red flesh watermelon (a singlemale sterile, “MS”, inbred plant of this genotype was selected) andSVR14227703 is a hybrid developed from the cross of WSB-142-1603 byBR618. Two hundred and fifty F2 individuals of theWSB-45-101S×SVR14227703 mapping population were transplanted for thecollection of phenotypic data on male sterility and genotyped withmultiple watermelon markers (Illumina GoldenGate OPA with 1202 validatedSNP markers). Tissue of the 250 F2 individuals and the parental linesfor genotyping was collected before transplanting the F2 plants in thefield.

The F2 individuals were transplanted with sufficient spacing to allowfor training of the vines and phenotyping at different time pointsduring the vine development. Individual plants were scored as malesterile or male fertile after evaluating the length of the flower bud'speduncle, the flower bud size, the presence or absence of anthers andpollen in open flowers, and differences in anther development in flowerbuds. Plants were evaluated at three different time points throughoutdevelopment to ensure that plants with male sterile flowers did notproduce male fertile flowers at a later stage, and phenotypic scoreswere found to be in agreement for all individuals of the F2 segregatingpopulation.

JoinMap 4 software was used to develop a linkage map of theWSB-45-101S×SVR14227703 mapping population and map ms-1 in thewatermelon genome. Briefly, linkage was calculated by independence LOD,ordering of linkage groups was conducted using the maximum likelihoodalgorithm, and genetic distances were estimated using the Haldanemapping function. The linkage map of WSB-45-101S×SVR14227703 consistedof 240 SNP markers, had a total of 20 linkage groups and map length of1514.3 cM, as compared to the 11 linkage groups of the consensus mapthat has a length of 1892.7 cM.

The ms-1 gene mapped on linkage group 2 of the consensus map in theinterval of the SNP markers NW0249314 (SEQ ID NO: 1)/NW0249599 (SEQ IDNO: 6) (colocalized) and NW0250301 (SEQ ID NO: 78). Due to the lack ofpolymorphic markers in this analysis in the interval ofNW0249314/NW0249599 and NW0250301, which spans 94.4 cM on the consensusmap, linkage group 2 split into two linkage groups inWSB-45-101S×SVR14227703. The most closely linked marker to ms-1 wasNW0250301 which is located 16.3 cM downstream of the locus on linkagegroup 2. The location of ms-1 was confirmed using QTL Cartographer. Asingle QTL peak was identified in the watermelon genome above the LODthreshold of 2.5 using Composite Interval Mapping analysis, and waslocated near the marker NW0250301 (LOD=30.5).

The marker assay data of diverse watermelon germplasm carrying the ms-1introgression was investigated for additional marker assays developed inthe interval between NW0249314/NW0249599 and NW0250301 (FIG. 1). For allthese lines, male sterility is maintained and the seed is harvested frommale sterile (ms-1, ms-1) and male fertile (Ms-1, ms-1) plants in a 1:1ratio. Consequently, these lines are expected to have heterozygousgenotypes at the ms-1 locus since bulk seed samples were used for markeranalysis. A region of heterozygosity was indeed identified for some ofthese lines near the SNP markers NW0248249 (SEQ ID NO: 22), NW0251340(SEQ ID NO: 53), and NW0248760 (SEQ ID NO: 64). This is not far from themost likely region of ms-1 gene estimated from inference of geneticdistance on the consensus map, which is highlighted in grey (FIG. 1).

Markers from this genomic region of linkage group 2 were selected togenotype a diverse panel of watermelon lines that were phenotyped on aplant basis. This panel includes 34 lines of 9 different market types(FIG. 1). Eight plants for each of these 34 lines that segregate formale sterility were transplanted in a randomized complete block (RCBD)design and tissue sampled. Phenotypic and genotypic data obtainedconfirmed marker-trait association in different market types andidentified an informative marker in the proximity of ms-1 thataccurately predicts the phenotype.

Example 2

A. Mapping

Using two F2 populations, WCS-146-21905/WNE-142-1204 (referred to aspopulation 1) and WSB-45-101S/WCS-39-100 (referred to as population 2),the ms-1 gene was mapped between 78 (NW0251130; SEQ ID NO: 23) and 101.8cM (NCLAN009104771; SEQ ID NO: 65). Using polymorphic markers in thegene region, a total of 1100 and 780 F2 plants were screened forrecombinants in the gene region from population 1 and 2, respectively. Atotal of 281 recombinants were selected and phenotyped for malesterility. Additional markers were developed and used to genotype theidentified recombinants. This analysis placed ms-1 between markersNW0251130 (SEQ ID NO: 23) and NCLAN009405170 (SEQ ID NO: 59) using atotal of 4 recombinants.

An additional 203 BC plants derived from 30 BC families (segregating formale sterility as in a backcross) were phenotyped and genotyped withmarkers in the ms-1 region to confirm its location (FIG. 2). Theanalysis indicated that ms-1 is located between NCLAN009404570 (SEQ IDNO: 28) and NCLAN009405170 (SEQ ID NO: 59). Three recombinants wereidentified between ms-1 and NCLAN009404570 (SEQ ID NO: 28) and sixrecombinants were identified between ms-1 and NCLAN009405170 (SEQ ID NO:59).

The data above shows that the ms-1 gene is located on LG 2 betweenNCLAN009404570 and NCLAN009405170.

B. Additional Fine Mapping

Two SNPs were converted into TaqMan markers (NCLAN009490491 [SEQ ID NO:43] and NCLAN009490296 [SEQ ID NO: 38]) and used for fine mapping. TheseSNPs were not present in all sterile families (only polymorphic in threefamilies). An indel (FIG. 3) was converted into the TaqMan markerNCLAN009584571 (SEQ ID NO: 33; 82.03 cM) for analysis on the finemapping panel and in a larger panel of sterile lines and elitegermplasm.

A total of 1223 plants from 5 BC1 families segregating for malesterility were analyzed with markers flanking the ms-1 gene. The markerswere NCLAN009404570 (SEQ ID NO: 28) and NCLAN009405170 (SEQ ID NO: 59)and 4 additional interstitial markers: NCLAN009490296 (SEQ ID NO: 38),NCLAN009490491 (SEQ ID NO: 43); NCLAN009491448 (SEQ ID NO: 48);NCLAN009490864 (SEQ ID NO: 54). A total of 74 recombinants wereidentified. Based on the distribution of the recombination events, 56recombinants were selected and phenotyped.

A total of 16 recombinants were identified between ms-1 andNCLAN009404570 (SEQ ID NO: 28) and 10 between ms-1 and NCLAN009491448.These recombinants strongly indicate that ms-1 is between NCLAN009404570(81 cM) and NCLAN009491448 (SEQ ID NO: 48; 82.46 cM) on LG2,corresponding to about 80 kb on chromosome 6.

After additional targeted re-sequencing, the mapping panel was analyzedwith the marker NCLAN009584571 (SEQ ID NO: 33). The marker was confirmedas being closely linked to ms-1, but did not reduce the interval of81-82.46 cM.

C. Accuracy Analysis

Regions in the interval containing ms-1 were targeted for re-sequencingusing the parents of the two mapping populations as well as fertile andsterile plants from five of the 30 male sterility families mentionedabove (segregating for male sterility as in backcross). The regions wereselected for re-sequencing using two criteria: (1) within a gene and/or(2) presenting informative SNP based on the whole genome re-sequencedlines (fixed in all 9 fertile lines with data and heterozygote in 1 linesegregating for male sterility). One single SNP, NCLAN009584571 (SEQ IDNO: 33) was found completely associated with the phenotype in there-sequencing panel.

An accuracy panel was assembled from a diverse set of 34 male sterileand 46 fertile lines. For the 34 male sterile families, eight plantsfrom each family were transplanted into the field for phenotyping ofindividual plants. Plants were phenotyped until there were at leastthree consistent ratings on sequential time points. TM markers in theinterval (81-82.5 cM) were run on the panel.

To analyze the male sterile lines it was first determined whether themarker was segregating with the phenotype, indicating if the marker willbe informative for differentiating male sterile and fertile plantswithin the family (% of families that marker is segregating; Table 2).The marker NCLAN009405170 (SEQ ID NO: 59) is segregating in all butthree of the families; however the marker NCLAN009584571 (SEQ ID NO: 33)is segregating in all families. Secondly, the fertile homozygous allelecalls were derived for each of the male sterile families. The frequencyof the sterile allele was then calculated in the sterile and fertilelines. The analysis of an accuracy panel showed that NCLAN009584571 ishighly associated with the ms-1 phenotype (Table 2).

TABLE 2 Allele frequency analysis for the presence of the sterile allelein sterile and fertile lines. Position (LG2) 81.02 82.03 82.05 84.2Group N Marker NCLAN009404570 NCLAN009584571 NCLAN009490296NCLAN009405170 Sterile (S) Allele WAS-45-2158S TT ** GG AA Sterile 34 Sallele freq 1.00 1.00 1.00 0.91 Fertile - FS 34 S allele freq 0.35 0.000.35 0.00 Fertile - elite 46 0.46 0.00 0.48 0.00 Fertile - all 80 0.410.00 0.43 0.00 % of FS families 34 0.65 1.00 0.68 0.91 that marker ishet

The ms-1 gene has been mapped to a 1.5 cM interval between 81-82.5 cM onLG2. The marker NCLAN009584571 (SEQ ID NO: 33) is highly associated tothe trait.

What is claimed is:
 1. A method of determining the genotype associatedwith a male sterile phenotype of a watermelon plant or part thereof, themethod comprising the steps of: obtaining a sample of nucleic acids fromsaid plant or part thereof; detecting in said sample at least a firstpolymorphism in or genetically linked to a locus that confers said malesterile phenotype, wherein said locus comprises a marker selected fromthe group consisting of NW0248489 (SEQ ID NO: 70), NCLAN009404570 (SEQID NO: 28), NCLAN009584571 (SEQ ID NO: 33), NCLAN009490296 (SEQ ID NO:38), NCLAN009491448 (SEQ ID NO: 48), NCLAN009490864 (SEQ ID NO: 54),NCLAN009405170 (SEQ ID NO: 59), and NW0248760 (SEQ ID NO: 64); andcrossing the plant comprising said first polymorphism with itself or adifferent plant to obtain at least a first progeny plant.
 2. A method ofidentifying a watermelon plant comprising a genotype associated with amale sterile phenotype, the method comprising the step of: (i) detectingin said plant at least a first polymorphism in or genetically linked toa locus that confers said male sterile phenotype comprising lociNW0249314 (SEQ ID NO: 1) and NW0250301 (SEQ ID NO: 78) on LG2; and (ii)selecting said watermelon plant from a population of watermelon plantsbased on the presence of said polymorphism.
 3. The method of claim 2,wherein said first polymorphism is selected from the group consistingof: NW0249314 (SEQ ID NO: 1), NW0249599 (SEQ ID NO: 6), NW0250496 (SEQID NO: 7), NW0249312 (SEQ ID NO: 12), NW0251153 (SEQ ID NO: 17),NW0248249 (SEQ ID NO: 22), NW0251130 (SEQ ID NO: 23), NCLAN009404570(SEQ ID NO: 28), NCLAN009584571 (SEQ ID NO: 33), NCLAN009490296 (SEQ IDNO: 38), NCLAN009490491 (SEQ ID NO: 43), NCLAN009491448 (SEQ ID NO: 48),NW0251340 (SEQ ID NO: 53), NCLAN009490864 (SEQ ID NO: 54),NCLAN009405170 (SEQ ID NO: 59), NW0248760 (SEQ ID NO: 64),NCLAN009104771 (SEQ ID NO: 65), NW0248489 (SEQ ID NO: 70), NW0249128(SEQ ID NO: 71), NW0251464 (SEQ ID NO: 72), NW0248953 (SEQ ID NO: 73),and NW0250301 (SEQ ID NO: 78).
 4. The method of claim 3, wherein saidfirst polymorphism is further defined as located in a genomic regionflanked by loci NCLAN009404570 (SEQ ID NO: 28) and NCLAN009405170 (SEQID NO: 59) on LG2.
 5. The method of claim 4, wherein said firstpolymorphism is NCLAN009584571 (SEQ ID NO: 33).
 6. A method forproducing a watermelon plant that comprises in its genome at least onelocus associated with a male sterile phenotype, the method comprising:(i) crossing a first watermelon plant and a second watermelon plant atleast one of which comprises a locus associated with a male sterilephenotype, wherein said locus comprises a marker selected from the groupconsisting of NW0248489 (SEQ ID NO: 70), NCLAN009404570 (SEQ ID NO: 28),NCLAN009584571 (SEQ ID NO: 33), NCLAN009490296 (SEQ ID NO: 38),NCLAN009491448 (SEQ ID NO: 48), NCLAN009490864 (SEQ ID NO: 54),NCLAN009405170 (SEQ ID NO: 59), and NW0248760 (SEQ ID NO: 64); (ii)detecting at least a first polymorphism in or genetically linked to saidlocus associated with a male sterile phenotype; and (iii) selecting awatermelon plant based on the presence of said polymorphism.
 7. Themethod of claim 6, further comprising the step of: (iv) crossing thewatermelon plant of step (iii) with itself or another watermelon plantto produce a further generation.
 8. The method of claim 7, furthercomprising the step of: (v) selecting a watermelon plant from thefurther generation based on the presence of said polymorphism.
 9. Themethod of claim 8, wherein steps (iii)-(v) are repeated from about 3times to about 10 times.
 10. The method of claim 6, wherein said firstpolymorphism is selected from the group consisting of: NW0249314 (SEQ IDNO: 1), NW0249599 (SEQ ID NO: 6), NW0250496 (SEQ ID NO: 7), NW0249312(SEQ ID NO: 12), NW0251153 (SEQ ID NO: 17), NW0248249 (SEQ ID NO: 22),NW0251130 (SEQ ID NO: 23), NCLAN009404570 (SEQ ID NO: 28),NCLAN009584571 (SEQ ID NO: 33), NCLAN009490296 (SEQ ID NO: 38),NCLAN009490491 (SEQ ID NO: 43), NCLAN009491448 (SEQ ID NO: 48),NW0251340 (SEQ ID NO: 53), NCLAN009490864 (SEQ ID NO: 54),NCLAN009405170 (SEQ ID NO: 59), NW0248760 (SEQ ID NO: 64),NCLAN009104771 (SEQ ID NO: 65), NW0248489 (SEQ ID NO: 70), NW0249128(SEQ ID NO: 71), NW0251464 (SEQ ID NO: 72), NW0248953 (SEQ ID NO: 73),and NW0250301 (SEQ ID NO: 78).
 11. The method of claim 10, wherein saidfirst polymorphism is located in or genetically linked to a genomicregion defined by loci NCLAN009404570 (SEQ ID NO: 28) and NCLAN009405170(SEQ ID NO: 59) on LG2; or within 15 cM thereof.
 12. The method of claim11, wherein said first polymorphism is NCLAN009584571 (SEQ ID NO: 33).13. A method of introgressing an allele into a watermelon plant, themethod comprising: (i) genotyping at least one watermelon plant in apopulation with respect to at least one polymorphism located in orgenetically linked to an allele that confers a male sterile phenotype,wherein said allele comprises a marker selected from the groupconsisting of NW0248489 (SEQ ID NO: 70), NCLAN009404570 (SEQ ID NO: 28),NCLAN009584571 (SEQ ID NO: 33), NCLAN009490296 (SEQ ID NO: 38),NCLAN009491448 (SEQ ID NO: 48), NCLAN009490864 (SEQ ID NO: 54),NCLAN009405170 (SEQ ID NO: 59), and NW0248760 (SEQ ID NO: 64); (ii)selecting from the population at least one watermelon plant comprisingsaid allele; and (iii) producing a population of plant or seedscomprising said allele.
 14. The method of claim 13, wherein thepolymorphism is selected from the group consisting of: NW0249314 (SEQ IDNO: 1), NW0249599 (SEQ ID NO: 6), NW0250496 (SEQ ID NO: 7), NW0249312(SEQ ID NO: 12), NW0251153 (SEQ ID NO: 17), NW0248249 (SEQ ID NO: 22),NW0251130 (SEQ ID NO: 23), NCLAN009404570 (SEQ ID NO: 28),NCLAN009584571 (SEQ ID NO: 33), NCLAN009490296 (SEQ ID NO: 38),NCLAN009490491 (SEQ ID NO: 43), NCLAN009491448 (SEQ ID NO: 48),NW0251340 (SEQ ID NO: 53), NCLAN009490864 (SEQ ID NO: 54),NCLAN009405170 (SEQ ID NO: 59), NW0248760 (SEQ ID NO: 64),NCLAN009104771 (SEQ ID NO: 65), NW0248489 (SEQ ID NO: 70), NW0249128(SEQ ID NO: 71), NW0251464 (SEQ ID NO: 72), NW0248953 (SEQ ID NO: 73),and NW0250301 (SEQ ID NO: 78).
 15. The method of claim 14, wherein thepolymorphism is located in a genomic region flanked by lociNCLAN009404570 (SEQ ID NO: 28) and NCLAN009405170 (SEQ ID NO: 59) onLG2; or within 15 cM thereof.
 16. The method of claim 15, wherein thepolymorphism is NCLAN009584571 (SEQ ID NO: 33).